Abstract Significant health relevance of this application is evident in our mechanistic dissection of the role of the cytoskeletal protein dystrophin in heart health and disease. Numerous inherited and acquired cardiac diseases are caused by deficits in dystrophin, including, notably, Duchenne muscular dystrophy (DMD), in which there is the complete loss of the dystrophin protein. Dystrophin is a 427 kDa cytoskeletal protein and is a vital link between the cytoskeleton, the muscle membrane and the extracellular matrix. Heart disease accounts for a significant mortality in DMD, for which there is no cure or long-term effective treatment. Recent advances in genetic technologies have fueled enthusiasm for gene-based therapeutic restitution of truncated dystrophins as a treatment for DMD. For DMD patients, several exon skipping clinical trial studies are ongoing and proof-of- concept somatic cell gene editing studies provide evidence of effectiveness in animal models. To date, none of these efforts have successfully translated to clinical efficacy in DMD patients. We posit reduced in vivo stability of internally truncated dystrophins is a significant barrier to ultimate clinical efficacy. We provide preliminary evidence that truncated dystrophins can be highly unstable in vivo, with markedly faster turnover rates than full length dystrophin. Reduced stability of a truncated dystrophin is expected to have significant implications for long-term clinical success by negatively impacting duration of therapeutic action in vivo. Further, the therapeutic expression threshold to confer significant cardio-protection with truncated dystrophin molecules is not known and truncated dystrophin proteins can confer only partial physiological restitution in dystrophin-deficient hearts in vivo. We have established a proof-of-concept approach to directly determine the stability and physiological expression threshold for truncated dystrophins in the heart in vivo. Guiding hypothesis: Clinically-designed gene therapy, gene-edited or exon skipped truncated dystrophin molecules will have significantly shorter half-lives in the heart in vivo, compared to intact full length dystrophin, compromising duration of action effects. The Specific Aims are to establish the in vivo stability/half-life of clinically relevant truncated dystrophins in the dystrophic heart. This knowledge is essential for the success of gene-based clinical trials for DMD. These studies will have a lasting impact on the field by illuminating the key dystrophin structure-function benchmarks required for long- term effectiveness of current and future gene-based therapies for DMD patients.